(W1030-06-34) Stabilization of a Therapeutic Peptide for a Quantitative ECLIA Assay

Purpose: A quantitative electrochemiluminescence immunoassay (ECLIA) assay was developed to determine a therapeutic peptide (46 amino acids) in human plasma stabilized with ethylenediaminetetraacetic acid (EDTA). The accuracy and precision were within the acceptance criteria with the initial method. However, the matrix effect with individual lots of normal human plasma (EDTA) showed that both LLOQ and High C were not accepted because of either over or underestimation. Investigation of the primary amino acid sequence of the peptide suggested the protein may be susceptible to proteolytic degradation. Therefore, protease inhibitors were tested to stabilize the therapeutic peptide in the matrix in order to improve the matrix effect of the assay.

Methods: For the determination of a therapeutic peptide in human plasma (EDTA), the therapeutic peptide in samples was pulled down onto a capturing regent coated MSD plate. After washing the plate, the bound therapeutic peptide was detected with the detection reagent followed by response readout using an electrochemiluminescence plate reader. For stabilizing the therapeutic peptide in human plasma (EDTA), both pooled and individual human plasma were spiked with protease inhibitors. For matrix effect testing, individual lots of human plasma (EDTA) from healthy volunteers were spiked with LLOQ and High QC level. The matrix effect samples were prepared with and without protease inhibitors. Each sample was divided into two aliquots. One aliquot was stored at -80°C right after aliquoting and the other was incubated at 25°C for 2 hours before being stored at -80°C. The prepared matrix effect samples were analyzed with the standards and QCs prepared with the pooled plasma spiked with protease inhibitors.

Results: There was no impact on accuracy and precision of the assay with spiking protease inhibitors into the pooled matrix before preparing standards and QCs. However, spiking protease inhibitors into individual lots of human plasma (EDTA) significantly improved the matrix effect of the sample. Of the 6 lots of individual human plasma (EDTA) tested for matrix effect during method development without spiking protease inhibitors, only 2 ME LLOQ samples were with acceptance  and none of ME High QC samples passed acceptance. While with spiking protease inhibitors, all 6 LLOQ samples and 5 High QC samples were accepted. Furthermore, sample stability was improved by protease inhibitors. After 2-hour incubation at 25°C, the mean concentration detected in ME LLOQ without protease inhibitors was decreased from 7.89 to 4.85 pg/mL, and the mean concentration of ME High QC without protease inhibitors was decreased from 277 to 182 pg/mL. In contrast, the mean concentrations detected in samples with protease inhibitors after 2-hour incubation at 25°C were 15.2 and 732 pg/mL for ME LLOQ and ME High QC, respectively, which were similar to the mean concentrations detected before incubation, 16.0 pg/mL for ME LLOQ, and 766 pg/mL for ME High QC.

Conclusion:

Spiking protease inhibitors into samples successfully improved the matrix effect of the assay to determine a therapeutic peptide in human plasma (EDTA). Furthermore, utilizing protease inhibitors may be more broadly useful for improving assay performance involving proteins that may be susceptible to proteolytic degradation.

Table 1 _ Improved Matrix Effect with Protease Inhibitors

Table 2 _ Improved Sample Stability with Protease Inhibitors